Abstract Millions of people worldwide are afflicted with sickle cell disease (SCD), a hereditary blood disease caused by a mutation of the -hemoglobin gene. Once thought to be simply due to increased rigidity of sickle red cells, SCD pathophysiology is now known to involve numerous cellular interactions among sickle red cells, white cells, platelets, reticulocytes (immature red cells), endothelial cells, and soluble factors (e.g. cytokines, coagulation factors, etc). Each of these pathologic interactions then contributes to microvascular occlusion, or vaso-occlusion, hemolysis (increased red destruction), and endothelial dysfunction. These key findings were primarily observed using in vivo animal models, which provide physiologically relevant but ultimately qualitative data. As SCD cellular interactions are inherently biophysical in nature, involving pathologic alterations in cell deformability and cell adhesion under hemodynamic conditions, quantitative methods are needed to fully understand the underlying mechanisms of these phenomena. Moreover, due to lack of sufficient technology, the relative contribution of each of these specific interactions on vaso-occlusion, hemolysis, and endothelial dysfunction are unknown. To that end, we have recently reported a novel endothelialized microfluidic in vitro model of the microvasculature that recapitulates and integrates this ensemble of pathophysiological processes at the physiologically relevant microvascular (<30 m) size scale (Tsai et al, JCI, 2012). This microvasculature-on-a-chip is ideally suited for the systematic and quantitative biophysical analyses of sickle cell vaso-occlusion, hemolysis, and endothelial dysfunction. For this work, we will further develop and optimize our microvasculature-on-a-chip microfluidic system to test our general hypothesis that in SCD, specific cellular interactions have differential effects on vaso-occlusion, hemolysis and endothelial dysfunction, and these contributions will change under different conditions, (e.g., white blood cell count, hemodynamics, anatomic site, on therapy, etc.). Specifically, we will first further optimize and build upon our system to enable the simultaneous testing of multiple conditions including concentrations of specific blood cell subpopulations, pharmacologic agents, wall shear stress, endothelial cell type, and oxygen tension, a major effector of sickle cell vaso-occlusion; this will achieve the high-throughput efficiency required to obtain the large amounts of data we need to extract from each experiment. We will then quantify the contributions of these cellular interactions under a variety of conditions to determine which dominate, and are therefore the most clinically relevant targets. Finally, we will apply varying dosages of standard and novel therapeutic agents to quantify their effects on those specific cellular interactions and to establish our microfluidic device as a viable drug discovery system for SCD. Overall, these studies will create a parameter space for the multitude of cell interactions in SCD and provide a quantitative framework for clinical hematologists to generate hypotheses and rationally design clinical trials for future SCD therapeutics.